*Target (protein/gene name):acidocalcisomal exopolyphosphatase *NCBI Gene # or RefSeq#:LMJF_01_0310
*Protein ID (NP or XP #) or Wolbachia#:29931
*Organism (including strain):Leishmania major strain Friedlin
Etiologic Risk Group (see link below): N/A
*/Disease Information (sort of like the Intro to your Mini Research Write up): Sometimes mistaken with leprosy, the affliction known as leishmaniosis, causes skin ulcers to erupt and may also lead to an enlarged liver and spleen – eventually leading to anemia. The disease is spread by the bite of sandflies, but the real culprit is the parasite protozoon belonging to the genus Leishmania; there are many species of this genus that cause the disease. Currently, about 12 million people are affected, with 20 to 50 thousand deaths per year.
Different species need to be treated differently. For examples, Leishmania major, is resistant to the oral medication Miltefosine; however, Pentamidine is effective.
--- List cost and quantity of substrate reagents, supplier, and catalog #
Fetal bovine serum: Cost must be inquired, Sigma, F2442 Dulbecco’s PBS: $15.40 for 100ML, Sigma, D1408 Protease inhibitor mixture: $64.10 for 1ML, Sigma, P8849 Protease inhibitor mixture: 71.80 for 1ML, Sigma, P8340 Ampicillin: $63.80 for 5G, Sigma, A9393 Kanamycin: $56.30 for 20ML, Sigma, K0254 Paraformaldehyde: $34.80 for 500G, Sigma, P6148 Glutaraldehyde: $116.50 for 10X1ML, Sigma, G5882 Bovine serum albumin: $276.00 for 50ML, Sigma, A7979 ATP: $293.50 for 1VL, Sigma, FLAAM PPi: $56.40 for 1VL, Sigma, P7275 phosphate glass: $121.50 for 500MG, Aldrich, S4379 proteinase K: $39.50 for 5MG, Aldrich, P2308 lysozyme: $49.50 for 1G, Sigma, L6876 horseradish peroxidase-conjugated anti-rabbit: $403.50 for 200UL, Sigma, H0912 fluorescein-conjugated goat anti-rabbit: $125.00 for 1ML, Sigma, F6005 rhodamine-conjugated goat anti-mouse IgGs: $178.50 for 1ML, Sigma, A0168
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 4ITY
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage: 99%
---- Max % Identities: 93%
---- Max Score: 1700
---- Total Score: 1700
Current Inhibitors: None
Expression Information (has it been expressed in bacterial cells): Expressed in Salmonella enterica, E. coli.
Purification Method: From: http://www.sciencedirect.com/science/article/pii/S0014579398005912 The vacuolar fraction free of other cell organelles [8]was frozen at −20°C in 10 mM Tris-HCl, pH 7.2, 10% glycerol, 0.5 mM phenylmethylsulfonyl fluoride and after thawing treated with ultrasound (MSE, USA) for 10 s. Vacuolar membranes were sedimented at 15 000×g for 90 min. The supernatant was applied to a Q-Sepharose column (1.6×5 cm) equilibrated with 25 mM Tris-acetate, pH 7.2, containing 0.1% Triton X-100 as enzyme stabilizer. After washing with the same buffer, extraneous proteins were eluted with the same buffer, containing 0.3 M KCl. PolyPase was eluted with 0.7 M KCl in the same buffer. Removal of KCl and substitution of the buffer with 25 mM Tris-acetate, pH 6.0, was performed in an Amicon system (PM-10 membrane). Thereafter, the preparation was applied to an S-Sepharose column (1.6×5 cm) equilibrated with 25 mM Tris-acetate, pH 6.0, containing 0.1% Triton X-100. Extraneous proteins were eluted with the same buffer, containing 0.2 M KCl. PolyPase was eluted with increasing concentrations of KCl (0.2–1.2 M) in the same buffer. The enzyme preparation was stored at −20°C.
Image of protein (PyMol with features delineated and shown separately): *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
*length of your protein in Amino Acids:388 Amino Acids
Molecular Weight of your protein in kiloDaltons using the**Expasy ProtParam**website:42.5948 kilo Daltons
MolarExtinction coefficient of your protein at 280 nm wavelength: Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water. Ext. coefficient 26525 Abs 0.1% (=1 g/l) 0.623, assuming all pairs of Cys residues form cystines Ext. coefficient 25900 Abs 0.1% (=1 g/l) 0.608, assuming all Cys residues are reduced
*NCBI Gene # or RefSeq#:LMJF_01_0310
*Protein ID (NP or XP #) or Wolbachia#:29931
*Organism (including strain):Leishmania major strain Friedlin
Etiologic Risk Group (see link below): N/A
*/Disease Information (sort of like the Intro to your Mini Research Write up):
Sometimes mistaken with leprosy, the affliction known as leishmaniosis, causes skin ulcers to erupt and may also lead to an enlarged liver and spleen – eventually leading to anemia. The disease is spread by the bite of sandflies, but the real culprit is the parasite protozoon belonging to the genus Leishmania; there are many species of this genus that cause the disease. Currently, about 12 million people are affected, with 20 to 50 thousand deaths per year.
Different species need to be treated differently. For examples, Leishmania major, is resistant to the oral medication Miltefosine; however, Pentamidine is effective.
Link to TDR Targets page (if present):http://www.tdrtargets.org/targets/view?gene_id=29931
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
Essentiality of this protein: http://www.ncbi.nlm.nih.gov/gene/12983123
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): multimer
Complex of proteins?: Monomer (chains a and b)
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): N/A
*EC#: 3.6.1.11
Link to BRENDA EC# page:http://www.brenda-enzymes.org/enzyme.php?ecno=3.6.1.11
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assayinformation (spectrophotometric, coupled assay ?, reagents):
-- link to Sigma (or othercompany) page for assay (see Sigma links below)http://www.sigmaaldrich.com/catalog/papers/12393865
--- List cost and quantity of substrate reagents, supplier, and catalog #
Fetal bovine serum: Cost must be inquired, Sigma, F2442
Dulbecco’s PBS: $15.40 for 100ML, Sigma, D1408
Protease inhibitor mixture: $64.10 for 1ML, Sigma, P8849
Protease inhibitor mixture: 71.80 for 1ML, Sigma, P8340
Ampicillin: $63.80 for 5G, Sigma, A9393
Kanamycin: $56.30 for 20ML, Sigma, K0254
Paraformaldehyde: $34.80 for 500G, Sigma, P6148
Glutaraldehyde: $116.50 for 10X1ML, Sigma, G5882
Bovine serum albumin: $276.00 for 50ML, Sigma, A7979
ATP: $293.50 for 1VL, Sigma, FLAAM
PPi: $56.40 for 1VL, Sigma, P7275
phosphate glass: $121.50 for 500MG, Aldrich, S4379
proteinase K: $39.50 for 5MG, Aldrich, P2308
lysozyme: $49.50 for 1G, Sigma, L6876
horseradish peroxidase-conjugated anti-rabbit: $403.50 for 200UL, Sigma, H0912
fluorescein-conjugated goat anti-rabbit: $125.00 for 1ML, Sigma, F6005
rhodamine-conjugated goat anti-mouse IgGs: $178.50 for 1ML, Sigma, A0168
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 4ITY
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage: 99%
---- Max % Identities: 93%
---- Max Score: 1700
---- Total Score: 1700
Current Inhibitors: None
Expression Information (has it been expressed in bacterial cells): Expressed in Salmonella enterica, E. coli.
Purification Method:
From: http://www.sciencedirect.com/science/article/pii/S0014579398005912
The vacuolar fraction free of other cell organelles [8]was frozen at −20°C in 10 mM Tris-HCl, pH 7.2, 10% glycerol, 0.5 mM phenylmethylsulfonyl fluoride and after thawing treated with ultrasound (MSE, USA) for 10 s. Vacuolar membranes were sedimented at 15 000×g for 90 min. The supernatant was applied to a Q-Sepharose column (1.6×5 cm) equilibrated with 25 mM Tris-acetate, pH 7.2, containing 0.1% Triton X-100 as enzyme stabilizer. After washing with the same buffer, extraneous proteins were eluted with the same buffer, containing 0.3 M KCl. PolyPase was eluted with 0.7 M KCl in the same buffer. Removal of KCl and substitution of the buffer with 25 mM Tris-acetate, pH 6.0, was performed in an Amicon system (PM-10 membrane). Thereafter, the preparation was applied to an S-Sepharose column (1.6×5 cm) equilibrated with 25 mM Tris-acetate, pH 6.0, containing 0.1% Triton X-100. Extraneous proteins were eluted with the same buffer, containing 0.2 M KCl. PolyPase was eluted with increasing concentrations of KCl (0.2–1.2 M) in the same buffer. The enzyme preparation was stored at −20°C.
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
<span style="color: #4f81bd;"> 1__0__ 2__0__ 3__0__ 4__0__ 5__0__ 6__0__ MSGVINDFLR RCLKKVAGKV QPLTVVQGNE GGDMDSIVGC IYLAMLFDKQ PKFGFENPVP 7__0__ 8__0__ 9__0__ 10__0__ 11__0__ 12__0__ ALNFPQEDFG LRNDVTNLFK ELGIDASLLM SVQRGQIAHN LVDIAALNAS VVLYDHNKLR 13__0__ 14__0__ 15__0__ 16__0__ 17__0__ 18__0__ ENQSDLASRV VGVVDHHFDE QQYLKTASKL RVLRTVGSAC TLVTELYREC GEDVVCPTLL 19__0__ 20__0__ 21__0__ 22__0__ 23__0__ 24__0__ TAPIVLDTVN FEPAQKKVTP EDIAAYEWLR AKEVADSADA AALFEKLSKW KDDVLALSVP 25__0__ 26__0__ 27__0__ 28__0__ 29__0__ 30__0__ QILRRDYKQF SFKARTQKGV MSAGTSSVPC ACKQLEAHFS VDLIVAEAAK YVEQHQLDVL 31__0__ 32__0__ 33__0__ 34__0__ 35__0__ 36__0__ IVAFAGKVGG KHTREVAFCA KPDVISFFAP FVAEAPDGVS FTVITKCQTV DGSYEYASYS 37__0__ 38__0__ LSDPSISRKK LVPALSEFLA EGTRSLCE </span>*length of your protein in Amino Acids:388 Amino Acids
Molecular Weight of your protein in kiloDaltons using the**Expasy ProtParam**website:42.5948 kilo Daltons
MolarExtinction coefficient of your protein at 280 nm wavelength:
Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.
Ext. coefficient 26525 Abs 0.1% (=1 g/l) 0.623, assuming all pairs of Cys residues form cystines
Ext. coefficient 25900 Abs 0.1% (=1 g/l) 0.608, assuming all Cys residues are reduced
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDSGene Sequence(paste as text only):
ATGTCTGGCGTTATCAACGACTTCTTGCGCCGCTGCCTCAAGAAGGTTGCCGGCAAAGTGCAGCCACTGA CCGTGGTGCAGGGAAACGAGGGAGGTGACATGGACAGCATTGTCGGCTGCATTTACCTCGCCATGCTTTT TGATAAGCAGCCCAAATTCGGTTTTGAGAACCCTGTGCCGGCGCTGAACTTTCCACAAGAAGATTTCGGC CTGCGCAACGATGTGACGAATCTCTTCAAGGAGCTCGGGATCGATGCATCGCTCTTGATGTCGGTACAGA GGGGACAGATCGCGCATAACCTTGTCGATATTGCTGCTCTCAACGCTTCGGTTGTTCTTTATGATCACAA TAAGCTTAGGGAGAACCAAAGCGACCTCGCCTCTAGAGTTGTCGGAGTAGTGGACCATCACTTTGACGAG CAGCAGTACCTCAAAACCGCATCCAAGCTAAGAGTTCTGCGAACTGTCGGATCCGCGTGCACACTCGTTA CGGAGCTCTACCGCGAATGTGGCGAGGATGTTGTGTGTCCCACGCTGCTAACGGCTCCGATTGTTCTGGA TACAGTGAACTTTGAGCCGGCACAAAAGAAGGTGACACCTGAGGATATCGCTGCATACGAGTGGTTGCGT GCAAAGGAGGTCGCTGACAGTGCTGACGCCGCGGCTCTCTTTGAAAAGCTGTCGAAGTGGAAGGATGACG TGCTGGCACTCAGCGTTCCACAAATCTTGAGGCGAGATTACAAACAGTTCAGCTTCAAAGCCAGAACGCA GAAGGGCGTTATGTCCGCGGGCACCAGTAGTGTGCCGTGTGCGTGCAAGCAGCTAGAAGCTCACTTTTCT GTGGATTTAATTGTGGCGGAGGCTGCCAAGTACGTGGAGCAGCATCAGTTGGATGTGCTCATCGTTGCAT TTGCCGGGAAAGTCGGCGGCAAGCACACTCGCGAGGTCGCCTTCTGTGCAAAGCCTGACGTGATCTCCTT TTTTGCTCCTTTTGTAGCGGAGGCCCCGGATGGCGTGTCATTTACCGTGATCACAAAGTGTCAGACGGTG GACGGATCATACGAGTACGCCTCGTACTCCCTCAGCGACCCTTCTATTTCTCGCAAGAAGCTGGTTCCAG CTCTGTCCGAATTCCTTGCCGAGGGGACACGGAGTCTTTGTGAGTAA
*GC% Content for gene: N/A
*CDS Gene Sequence (codon optimized) - copy fromoutputofPrimer DesignProtocol (paste as text only): N/A
*GC% Content for gene (codon optimized): N/A